Introduction
Project Purpose and Significance
Obesity continues to increase as a public health emergency, with the origins of most adult obesity being in childhood. Effective clinical approaches are urgently needed to prevent or reverse childhood obesity. The gut-associated microbiome is an established central factor in energy harvest, hepatic function, insulin sensitivity, and adipose tissue homeostasis, making it a critical target for obesity intervention strategies.
This project represents a collaborative effort between Lurie Children’s Hospital and Abbott Nutrition to investigate interpersonal variation in energetics and short-chain fatty acid (SCFA) production of obesity-associated gut microbiota in response to slow and fast digestible carbohydrates. The work builds upon foundational research demonstrating that carbohydrate quality, rather than quantity alone, plays a crucial role in metabolic health.
Scientific Foundation and Motivation
The project was motivated by compelling evidence from the Frontiers in Nutrition paper by Wang et al. (2022) that demonstrated the therapeutic potential of slowly digestible carbohydrates (SDC) in metabolic syndrome and obesity management. This seminal work showed that SDC displays beneficial effects on reducing glucose excursions in healthy, insulin-resistant, and type 2 diabetic individuals, inducing a slow and prolonged glucose release that results in reduced postprandial glycemic responses and extended glycemic index values.
In type 2 diabetic patients, SDC-rich diets (60g/day) reduced glycemic variability parameters by 17-23%, with these parameters correlating with HbA1c, suggesting potential for long-term glycemic improvement. The Frontiers paper also demonstrated that foods with the highest SDC content (23.9-27.5 g/100g) induce the lowest glycemic responses with the lowest incremental AUC of glucose and insulin concentration.
Comparative Evidence from Murine Models
In comparison to fast digestible carbohydrate (FDC) sources, the Abbott group has shown in murine models of obesity that nutrition with slow digestible carbohydrates (SDC) reverses obesity-associated phenotypes, including elevated body mass, insulin resistance, and systemic inflammation. However, the interpersonal differences in SDC responses by the childhood-associated human microbiota may not be fully predicted in murine obesity models.
Interpersonal Variation in Human Microbiota
Lurie investigators and colleagues have shown interpersonal variation in the production of short-chain fatty acids (SCFA) by the human gut microbiome from adolescents with obesity in response to ex vivo prebiotic exposure, suggesting that complex carbohydrate utilization by the microbiota varies between individuals and thus may affect who responds to SDC and other nutritional approaches to obesity.
Understanding the variation in the compositional and metabolic responses of the childhood-associated microbiota may inform future obesity-treatment trials and precision approaches to obesity therapy.
Project Objectives
The main objectives of this project are to:
- Measure variation in responses between different human gut-associated microbiome communities to FDC and SDC
- Identify childhood-associated organisms with facile utilization of SDC
- Test interindividual variation of short-chain fatty acid production among fecal microbiota samples to slow and fast digestible carbohydrates
- Measure energy harvest differences between human obesity-associated fecal microbiota
- Isolate SDC bacterial utilizers using single cell isolation techniques towards the future goal of creating obesity treatment synbiotic combinations
Clinical Impact
Understanding the interpersonal differences in SDC utilization by the childhood-associated human microbiota may inform future obesity-treatment trials through the identification of likely responders and, subsequently, precision approaches to obesity therapy. This precision nutrition approach could revolutionize childhood obesity treatment by enabling personalized dietary interventions based on individual microbiome composition and metabolic capacity.
Analysis Overview
This document presents the analysis of short-chain fatty acids (SCFAs) in the Abbott carbohydrate obesity project. The analysis examines how SCFA analytes change between experimental groups, carbohydrate types, and time points, with particular focus on identifying interpersonal variation in metabolic responses to different carbohydrate sources.
Methods
SCFA Analysis using PFBBr Panel
Short chain fatty acids were analyzed using Gas chromatography-mass spectrometry (GC-MS) following derivatization with pentafluorobenzyl bromide (PFBBr). SCFAs (acetate, butyrate, propionate) were quantitatively analyzed following PFB derivatization and detection by negative collision induced gas chromatography-mass spectrometry ((-)-CI-GC-MS, Agilent 8890). Additional compounds including 5-aminovalerate and succinate were also quantified.
Detailed SCFA Analysis Protocol
The following section outlines the specific protocol used for SCFA derivatization and GC-MS analysis.
Short chain fatty acids were derivatized as described by Haak et al. with modifications. The metabolite extract (100 µL) was added to 100 µL of 100 mM borate buffer (pH 10), 400 µL of 100 mM pentafluorobenzyl bromide in Acetonitrile, and 400 µL of n-hexane in a capped mass spec autosampler vial. Samples were heated to 65°C for 1 hour while shaking at 1300 rpm. After cooling, samples were centrifuged at 4°C, 2000 x g for 5 min, allowing phase separation. The hexanes phase was transferred and analyzed.
Samples were analyzed using a GC-MS (Agilent 7890A GC system, Agilent 5975C MS detector) operating in negative chemical ionization mode, using a HP-5MSUI column (30 m x 0.25 mm, 0.25 µm), methane as the reagent gas and 1 µL split injection (1:10 split ratio). A 10-point calibration curve was prepared with acetate (100 mM), propionate (25 mM), butyrate (12.5 mM), and succinate (50 mM), with 9 subsequent 2x serial dilutions.
Summary Statistics
## Total observations after averaging technical replicates: 480
Summary by Group
Summary Statistics by Experimental Group
|
Group
|
Analyte
|
n
|
Mean
|
Median
|
SD
|
SEM
|
Q25
|
Q75
|
|
Case
|
5aminovalerate
|
48
|
0.485
|
0.245
|
0.561
|
0.081
|
0.050
|
0.701
|
|
Control
|
5aminovalerate
|
48
|
0.554
|
0.495
|
0.504
|
0.073
|
0.050
|
0.959
|
|
Case
|
acetate
|
48
|
17.304
|
13.109
|
16.795
|
2.424
|
1.201
|
32.258
|
|
Control
|
acetate
|
48
|
18.734
|
22.513
|
16.704
|
2.411
|
1.214
|
33.208
|
|
Case
|
butyrate
|
48
|
3.569
|
1.635
|
3.786
|
0.546
|
0.260
|
7.062
|
|
Control
|
butyrate
|
48
|
4.245
|
3.745
|
4.214
|
0.608
|
0.291
|
7.284
|
|
Case
|
propionate
|
48
|
2.733
|
1.393
|
2.955
|
0.427
|
0.170
|
4.450
|
|
Control
|
propionate
|
48
|
2.750
|
2.963
|
2.614
|
0.377
|
0.184
|
4.782
|
|
Case
|
succinate
|
48
|
0.779
|
0.170
|
1.244
|
0.180
|
0.080
|
1.238
|
|
Control
|
succinate
|
48
|
1.243
|
0.345
|
1.722
|
0.249
|
0.132
|
1.788
|
Summary by Carbohydrate Type
Summary Statistics by Carbohydrate Type
|
Carbohydrate Type
|
Analyte
|
n
|
Mean
|
Median
|
SD
|
SEM
|
Q25
|
Q75
|
|
No Carbohydrate
|
5aminovalerate
|
32
|
0.560
|
0.488
|
0.554
|
0.098
|
0.050
|
0.895
|
|
Rapid Digestible
|
5aminovalerate
|
32
|
0.489
|
0.392
|
0.513
|
0.091
|
0.050
|
0.919
|
|
Slow Digestible
|
5aminovalerate
|
32
|
0.509
|
0.392
|
0.541
|
0.096
|
0.050
|
0.787
|
|
No Carbohydrate
|
acetate
|
32
|
16.048
|
17.312
|
14.812
|
2.618
|
1.250
|
30.038
|
|
Rapid Digestible
|
acetate
|
32
|
18.233
|
23.961
|
17.056
|
3.015
|
1.152
|
33.003
|
|
Slow Digestible
|
acetate
|
32
|
19.776
|
25.528
|
18.295
|
3.234
|
1.188
|
36.237
|
|
No Carbohydrate
|
butyrate
|
32
|
3.201
|
3.150
|
2.956
|
0.523
|
0.312
|
6.232
|
|
Rapid Digestible
|
butyrate
|
32
|
4.000
|
2.799
|
4.396
|
0.777
|
0.279
|
7.631
|
|
Slow Digestible
|
butyrate
|
32
|
4.519
|
4.089
|
4.476
|
0.791
|
0.282
|
7.884
|
|
No Carbohydrate
|
propionate
|
32
|
3.206
|
3.470
|
3.125
|
0.552
|
0.188
|
6.151
|
|
Rapid Digestible
|
propionate
|
32
|
2.244
|
2.411
|
2.244
|
0.397
|
0.168
|
3.516
|
|
Slow Digestible
|
propionate
|
32
|
2.775
|
2.407
|
2.885
|
0.510
|
0.189
|
4.630
|
|
No Carbohydrate
|
succinate
|
32
|
0.691
|
0.262
|
0.979
|
0.173
|
0.000
|
0.700
|
|
Rapid Digestible
|
succinate
|
32
|
1.067
|
0.255
|
1.570
|
0.278
|
0.115
|
1.341
|
|
Slow Digestible
|
succinate
|
32
|
1.275
|
0.245
|
1.846
|
0.326
|
0.114
|
1.974
|
Summary by Time Point
Summary Statistics by Time Point
|
Time (Hours)
|
Analyte
|
n
|
Mean
|
Median
|
SD
|
SEM
|
Q25
|
Q75
|
|
0
|
5aminovalerate
|
48
|
0.088
|
0.050
|
0.112
|
0.016
|
0.050
|
0.066
|
|
48
|
5aminovalerate
|
48
|
0.950
|
0.900
|
0.423
|
0.061
|
0.677
|
1.177
|
|
0
|
acetate
|
48
|
2.484
|
1.208
|
5.378
|
0.776
|
1.043
|
1.322
|
|
48
|
acetate
|
48
|
33.554
|
33.065
|
6.347
|
0.916
|
29.494
|
38.398
|
|
0
|
butyrate
|
48
|
0.546
|
0.272
|
0.949
|
0.137
|
0.230
|
0.413
|
|
48
|
butyrate
|
48
|
7.267
|
7.119
|
2.890
|
0.417
|
4.744
|
8.834
|
|
0
|
propionate
|
48
|
0.362
|
0.170
|
0.735
|
0.106
|
0.135
|
0.233
|
|
48
|
propionate
|
48
|
5.122
|
4.815
|
1.860
|
0.268
|
3.550
|
6.586
|
|
0
|
succinate
|
48
|
0.280
|
0.125
|
0.664
|
0.096
|
0.076
|
0.236
|
|
48
|
succinate
|
48
|
1.742
|
1.327
|
1.758
|
0.254
|
0.404
|
2.714
|
Combined Summary Statistics
## Combined summary table contains 60 condition combinations
## Combined summary saved to results/combined_summary_statistics.csv
Statistical Analysis
## Subject-level and case-only analyses saved to results/ directory
Statistical Results
We performed several statistical tests to compare SCFA concentrations across different experimental conditions. The following tables summarize the results of these comparisons, including t-tests for group differences and ANOVA for carbohydrate effects.
Group Comparisons (Control vs Case)
This table presents the results of t-tests comparing SCFA concentrations between the control and case groups.
Group Comparisons with Benjamini-Hochberg Correction
|
Analyte
|
.y.
|
group1
|
group2
|
n1
|
n2
|
statistic
|
df
|
P-value
|
Adjusted P-value
|
Significance
|
|
5aminovalerate
|
concentration
|
control
|
case
|
48
|
48
|
0.6290
|
92.9323
|
0.531
|
0.8463
|
ns
|
|
acetate
|
concentration
|
control
|
case
|
48
|
48
|
0.4183
|
93.9972
|
0.677
|
0.8463
|
ns
|
|
butyrate
|
concentration
|
control
|
case
|
48
|
48
|
0.8265
|
92.9395
|
0.411
|
0.8463
|
ns
|
|
propionate
|
concentration
|
control
|
case
|
48
|
48
|
0.0300
|
92.6226
|
0.976
|
0.9760
|
ns
|
|
succinate
|
concentration
|
control
|
case
|
48
|
48
|
1.5115
|
85.5443
|
0.134
|
0.6700
|
ns
|
Carbohydrate Type Comparisons
This table shows the results of ANOVA tests examining the effect of different carbohydrate types on SCFA concentrations.
ANOVA Results for Carbohydrate Type Effects
|
Analyte
|
Effect
|
DFn
|
DFd
|
F
|
P-value
|
p<.05
|
ges
|
Adjusted P-value
|
Significance
|
|
5aminovalerate
|
carbohydrate_type
|
2
|
93
|
0.147
|
0.864
|
|
0.003
|
0.8640
|
ns
|
|
acetate
|
carbohydrate_type
|
2
|
93
|
0.399
|
0.672
|
|
0.008
|
0.8400
|
ns
|
|
butyrate
|
carbohydrate_type
|
2
|
93
|
0.880
|
0.418
|
|
0.019
|
0.6967
|
ns
|
|
propionate
|
carbohydrate_type
|
2
|
93
|
0.965
|
0.385
|
|
0.020
|
0.6967
|
ns
|
|
succinate
|
carbohydrate_type
|
2
|
93
|
1.233
|
0.296
|
|
0.026
|
0.6967
|
ns
|
Post-hoc Carbohydrate Comparisons
Following the ANOVA, pairwise t-tests were performed to compare each carbohydrate type to the ‘no carbohydrate’ control. The results are shown below.
Pairwise Comparisons vs No Carbohydrate Control
|
Analyte
|
.y.
|
group1
|
group2
|
n1
|
n2
|
P-value
|
p.signif
|
Adjusted P-value
|
Significance
|
|
5aminovalerate
|
concentration
|
no_carbohydrate
|
rapid_digestible
|
32
|
32
|
0.601
|
ns
|
0.6711
|
ns
|
|
5aminovalerate
|
concentration
|
no_carbohydrate
|
slow_digestible
|
32
|
32
|
0.706
|
ns
|
0.7060
|
ns
|
|
acetate
|
concentration
|
no_carbohydrate
|
rapid_digestible
|
32
|
32
|
0.604
|
ns
|
0.6711
|
ns
|
|
acetate
|
concentration
|
no_carbohydrate
|
slow_digestible
|
32
|
32
|
0.377
|
ns
|
0.6711
|
ns
|
|
butyrate
|
concentration
|
no_carbohydrate
|
rapid_digestible
|
32
|
32
|
0.427
|
ns
|
0.6711
|
ns
|
|
butyrate
|
concentration
|
no_carbohydrate
|
slow_digestible
|
32
|
32
|
0.191
|
ns
|
0.6367
|
ns
|
|
propionate
|
concentration
|
no_carbohydrate
|
rapid_digestible
|
32
|
32
|
0.169
|
ns
|
0.6367
|
ns
|
|
propionate
|
concentration
|
no_carbohydrate
|
slow_digestible
|
32
|
32
|
0.535
|
ns
|
0.6711
|
ns
|
|
succinate
|
concentration
|
no_carbohydrate
|
rapid_digestible
|
32
|
32
|
0.322
|
ns
|
0.6711
|
ns
|
|
succinate
|
concentration
|
no_carbohydrate
|
slow_digestible
|
32
|
32
|
0.125
|
ns
|
0.6367
|
ns
|
Three-way Interaction Analysis
To assess the combined effects of group, carbohydrate type, and time, a three-way ANOVA was conducted. The results are summarized in this table.
Three-way ANOVA: Group × Carbohydrate × Time Interactions
|
Analyte
|
Effect
|
P-value
|
Adjusted P-value
|
Significance
|
|
5aminovalerate
|
group
|
0.300
|
0.6125
|
ns
|
|
5aminovalerate
|
carbohydrate_type
|
0.666
|
0.9212
|
ns
|
|
5aminovalerate
|
timepoint_hr
|
0.000
|
0.0000
|
****
|
|
5aminovalerate
|
group:carbohydrate_type
|
0.935
|
0.9680
|
ns
|
|
5aminovalerate
|
group:timepoint_hr
|
0.706
|
0.9212
|
ns
|
|
5aminovalerate
|
carbohydrate_type:timepoint_hr
|
0.731
|
0.9212
|
ns
|
|
5aminovalerate
|
group:carbohydrate_type:timepoint_hr
|
0.942
|
0.9680
|
ns
|
|
acetate
|
group
|
0.222
|
0.5180
|
ns
|
|
acetate
|
carbohydrate_type
|
0.036
|
0.1435
|
ns
|
|
acetate
|
timepoint_hr
|
0.000
|
0.0000
|
****
|
|
acetate
|
group:carbohydrate_type
|
0.440
|
0.8105
|
ns
|
|
acetate
|
group:timepoint_hr
|
0.315
|
0.6125
|
ns
|
|
acetate
|
carbohydrate_type:timepoint_hr
|
0.114
|
0.3069
|
ns
|
|
acetate
|
group:carbohydrate_type:timepoint_hr
|
0.737
|
0.9212
|
ns
|
|
butyrate
|
group
|
0.112
|
0.3069
|
ns
|
|
butyrate
|
carbohydrate_type
|
0.041
|
0.1435
|
ns
|
|
butyrate
|
timepoint_hr
|
0.000
|
0.0000
|
****
|
|
butyrate
|
group:carbohydrate_type
|
0.576
|
0.9164
|
ns
|
|
butyrate
|
group:timepoint_hr
|
0.658
|
0.9212
|
ns
|
|
butyrate
|
carbohydrate_type:timepoint_hr
|
0.038
|
0.1435
|
ns
|
|
butyrate
|
group:carbohydrate_type:timepoint_hr
|
0.571
|
0.9164
|
ns
|
|
propionate
|
group
|
0.951
|
0.9680
|
ns
|
|
propionate
|
carbohydrate_type
|
0.022
|
0.1283
|
ns
|
|
propionate
|
timepoint_hr
|
0.000
|
0.0000
|
****
|
|
propionate
|
group:carbohydrate_type
|
0.963
|
0.9680
|
ns
|
|
propionate
|
group:timepoint_hr
|
0.256
|
0.5600
|
ns
|
|
propionate
|
carbohydrate_type:timepoint_hr
|
0.027
|
0.1350
|
ns
|
|
propionate
|
group:carbohydrate_type:timepoint_hr
|
0.950
|
0.9680
|
ns
|
|
succinate
|
group
|
0.095
|
0.3023
|
ns
|
|
succinate
|
carbohydrate_type
|
0.218
|
0.5180
|
ns
|
|
succinate
|
timepoint_hr
|
0.000
|
0.0000
|
****
|
|
succinate
|
group:carbohydrate_type
|
0.960
|
0.9680
|
ns
|
|
succinate
|
group:timepoint_hr
|
0.659
|
0.9212
|
ns
|
|
succinate
|
carbohydrate_type:timepoint_hr
|
0.492
|
0.8610
|
ns
|
|
succinate
|
group:carbohydrate_type:timepoint_hr
|
0.968
|
0.9680
|
ns
|
Subject-Level Analyses
To account for individual variability, we conducted analyses at the subject level. This allows us to examine within-subject changes and summarize statistics for each participant, providing a more granular view of the data.
Subject-Level Summary Statistics
This table provides summary statistics for each subject, including the mean concentration and number of observations.
Subject-Level Summary Statistics
|
Group
|
Analyte
|
n Subjects
|
Mean Subject Means
|
SD Subject Means
|
SEM Subjects
|
|
control
|
5aminovalerate
|
8
|
0.554
|
0.124
|
0.044
|
|
control
|
acetate
|
8
|
18.734
|
5.296
|
1.872
|
|
control
|
butyrate
|
8
|
4.245
|
1.321
|
0.467
|
|
control
|
propionate
|
8
|
2.750
|
0.897
|
0.317
|
|
control
|
succinate
|
8
|
1.243
|
1.137
|
0.402
|
|
case
|
5aminovalerate
|
8
|
0.485
|
0.276
|
0.098
|
|
case
|
acetate
|
8
|
17.304
|
2.657
|
0.940
|
|
case
|
butyrate
|
8
|
3.569
|
1.103
|
0.390
|
|
case
|
propionate
|
8
|
2.733
|
0.999
|
0.353
|
|
case
|
succinate
|
8
|
0.779
|
0.707
|
0.250
|
Within-Subject Changes (0h to 48h)
This table shows the results of statistical tests on the changes in SCFA concentrations within each subject from baseline to 48 hours.
Within-Subject Changes from Baseline to 48h
|
Group
|
Carbohydrate Type
|
Analyte
|
n
|
Mean Change
|
SD Change
|
SEM Change
|
t-statistic
|
P-value
|
Adjusted P-value
|
Significance
|
|
control
|
no_carbohydrate
|
5aminovalerate
|
8
|
0.9675
|
0.4641
|
0.1641
|
5.8963
|
0.0006
|
0.0009
|
***
|
|
control
|
no_carbohydrate
|
acetate
|
8
|
26.0637
|
8.4143
|
2.9749
|
8.7612
|
0.0001
|
0.0001
|
***
|
|
control
|
no_carbohydrate
|
butyrate
|
8
|
4.8569
|
1.9468
|
0.6883
|
7.0564
|
0.0002
|
0.0003
|
***
|
|
control
|
no_carbohydrate
|
propionate
|
8
|
5.2625
|
1.4668
|
0.5186
|
10.1473
|
0.0000
|
0.0001
|
***
|
|
control
|
no_carbohydrate
|
succinate
|
8
|
1.2794
|
1.1714
|
0.4141
|
3.0892
|
0.0176
|
0.0657
|
ns
|
|
control
|
rapid_digestible
|
5aminovalerate
|
8
|
0.7974
|
0.2539
|
0.0898
|
8.8830
|
0.0000
|
0.0001
|
***
|
|
control
|
rapid_digestible
|
acetate
|
8
|
31.2600
|
6.8995
|
2.4393
|
12.8150
|
0.0000
|
0.0000
|
***
|
|
control
|
rapid_digestible
|
butyrate
|
8
|
7.3123
|
4.1611
|
1.4712
|
4.9704
|
0.0016
|
0.0017
|
**
|
|
control
|
rapid_digestible
|
propionate
|
8
|
3.4114
|
1.8180
|
0.6428
|
5.3073
|
0.0011
|
0.0011
|
**
|
|
control
|
rapid_digestible
|
succinate
|
8
|
1.5103
|
2.0037
|
0.7084
|
2.1320
|
0.0705
|
0.0705
|
ns
|
|
control
|
slow_digestible
|
5aminovalerate
|
8
|
0.8964
|
0.2789
|
0.0986
|
9.0918
|
0.0000
|
0.0001
|
***
|
|
control
|
slow_digestible
|
acetate
|
8
|
32.3582
|
8.0537
|
2.8474
|
11.3640
|
0.0000
|
0.0000
|
***
|
|
control
|
slow_digestible
|
butyrate
|
8
|
8.5557
|
3.3129
|
1.1713
|
7.3045
|
0.0002
|
0.0003
|
***
|
|
control
|
slow_digestible
|
propionate
|
8
|
4.6503
|
1.9143
|
0.6768
|
6.8711
|
0.0002
|
0.0004
|
***
|
|
control
|
slow_digestible
|
succinate
|
8
|
1.9625
|
2.2616
|
0.7996
|
2.4544
|
0.0438
|
0.0657
|
ns
|
|
case
|
no_carbohydrate
|
5aminovalerate
|
8
|
0.8937
|
0.3891
|
0.1376
|
6.4970
|
0.0003
|
0.0007
|
***
|
|
case
|
no_carbohydrate
|
acetate
|
8
|
29.9688
|
3.4826
|
1.2313
|
24.3397
|
0.0000
|
0.0000
|
***
|
|
case
|
no_carbohydrate
|
butyrate
|
8
|
5.7231
|
1.1415
|
0.4036
|
14.1811
|
0.0000
|
0.0000
|
***
|
|
case
|
no_carbohydrate
|
propionate
|
8
|
6.0431
|
1.8893
|
0.6680
|
9.0472
|
0.0000
|
0.0001
|
***
|
|
case
|
no_carbohydrate
|
succinate
|
8
|
0.8456
|
1.1009
|
0.3892
|
2.1726
|
0.0664
|
0.0705
|
ns
|
|
case
|
rapid_digestible
|
5aminovalerate
|
8
|
0.8112
|
0.5696
|
0.2014
|
4.0281
|
0.0050
|
0.0057
|
**
|
|
case
|
rapid_digestible
|
acetate
|
8
|
31.0581
|
6.9134
|
2.4442
|
12.7066
|
0.0000
|
0.0000
|
***
|
|
case
|
rapid_digestible
|
butyrate
|
8
|
6.5300
|
3.7671
|
1.3319
|
4.9029
|
0.0017
|
0.0017
|
**
|
|
case
|
rapid_digestible
|
propionate
|
8
|
4.1556
|
1.5547
|
0.5497
|
7.5602
|
0.0001
|
0.0003
|
***
|
|
case
|
rapid_digestible
|
succinate
|
8
|
1.4047
|
1.5458
|
0.5465
|
2.5702
|
0.0370
|
0.0657
|
ns
|
|
case
|
slow_digestible
|
5aminovalerate
|
8
|
0.8075
|
0.5815
|
0.2056
|
3.9275
|
0.0057
|
0.0057
|
**
|
|
case
|
slow_digestible
|
acetate
|
8
|
35.7078
|
5.2959
|
1.8724
|
19.0709
|
0.0000
|
0.0000
|
***
|
|
case
|
slow_digestible
|
butyrate
|
8
|
7.3503
|
2.2995
|
0.8130
|
9.0408
|
0.0000
|
0.0001
|
***
|
|
case
|
slow_digestible
|
propionate
|
8
|
5.0372
|
2.3603
|
0.8345
|
6.0363
|
0.0005
|
0.0006
|
***
|
|
case
|
slow_digestible
|
succinate
|
8
|
1.7725
|
1.7861
|
0.6315
|
2.8069
|
0.0263
|
0.0657
|
ns
|
Case-Only Temporal Analysis
To isolate the effects of the intervention within the case group, we performed a temporal analysis comparing SCFA concentrations at 0h and 48h. This analysis helps to understand the direct impact of the carbohydrate types on the case subjects over time.
Case Group Temporal Changes by Carbohydrate Type
This table presents the temporal changes in SCFA concentrations for the case group, broken down by carbohydrate type.
Case Group Only: Temporal Changes (0h to 48h) by Carbohydrate Type
|
Carbohydrate Type
|
Analyte
|
n
|
Mean Change
|
SD Change
|
SEM Change
|
t-statistic
|
P-value
|
Adjusted P-value
|
Significance
|
|
no_carbohydrate
|
5aminovalerate
|
8
|
0.8937
|
0.3891
|
0.1376
|
6.4970
|
0.0003
|
0.0010
|
**
|
|
no_carbohydrate
|
acetate
|
8
|
29.9688
|
3.4826
|
1.2313
|
24.3397
|
0.0000
|
0.0000
|
***
|
|
no_carbohydrate
|
butyrate
|
8
|
5.7231
|
1.1415
|
0.4036
|
14.1811
|
0.0000
|
0.0000
|
***
|
|
no_carbohydrate
|
propionate
|
8
|
6.0431
|
1.8893
|
0.6680
|
9.0472
|
0.0000
|
0.0001
|
***
|
|
no_carbohydrate
|
succinate
|
8
|
0.8456
|
1.1009
|
0.3892
|
2.1726
|
0.0664
|
0.0664
|
ns
|
|
rapid_digestible
|
5aminovalerate
|
8
|
0.8112
|
0.5696
|
0.2014
|
4.0281
|
0.0050
|
0.0057
|
**
|
|
rapid_digestible
|
acetate
|
8
|
31.0581
|
6.9134
|
2.4442
|
12.7066
|
0.0000
|
0.0000
|
***
|
|
rapid_digestible
|
butyrate
|
8
|
6.5300
|
3.7671
|
1.3319
|
4.9029
|
0.0017
|
0.0017
|
**
|
|
rapid_digestible
|
propionate
|
8
|
4.1556
|
1.5547
|
0.5497
|
7.5602
|
0.0001
|
0.0002
|
***
|
|
rapid_digestible
|
succinate
|
8
|
1.4047
|
1.5458
|
0.5465
|
2.5702
|
0.0370
|
0.0555
|
ns
|
|
slow_digestible
|
5aminovalerate
|
8
|
0.8075
|
0.5815
|
0.2056
|
3.9275
|
0.0057
|
0.0057
|
**
|
|
slow_digestible
|
acetate
|
8
|
35.7078
|
5.2959
|
1.8724
|
19.0709
|
0.0000
|
0.0000
|
***
|
|
slow_digestible
|
butyrate
|
8
|
7.3503
|
2.2995
|
0.8130
|
9.0408
|
0.0000
|
0.0001
|
***
|
|
slow_digestible
|
propionate
|
8
|
5.0372
|
2.3603
|
0.8345
|
6.0363
|
0.0005
|
0.0005
|
***
|
|
slow_digestible
|
succinate
|
8
|
1.7725
|
1.7861
|
0.6315
|
2.8069
|
0.0263
|
0.0555
|
ns
|
Case Group Temporal Changes (Pooled)
Here, we present the temporal changes for the case group, pooled across all carbohydrate types to assess the overall time effect.
Case Group Only: Temporal Changes (0h to 48h) Pooled Across Carbohydrate Types
|
Analyte
|
n
|
Mean Change
|
SD Change
|
SEM Change
|
t-statistic
|
P-value
|
Adjusted P-value
|
Significance
|
|
5aminovalerate
|
24
|
0.8375
|
0.4994
|
0.1019
|
8.2155
|
0e+00
|
0e+00
|
***
|
|
acetate
|
24
|
32.2449
|
5.7651
|
1.1768
|
27.4006
|
0e+00
|
0e+00
|
***
|
|
butyrate
|
24
|
6.5345
|
2.6049
|
0.5317
|
12.2894
|
0e+00
|
0e+00
|
***
|
|
propionate
|
24
|
5.0786
|
2.0342
|
0.4152
|
12.2310
|
0e+00
|
0e+00
|
***
|
|
succinate
|
24
|
1.3409
|
1.4895
|
0.3040
|
4.4104
|
2e-04
|
2e-04
|
***
|
Case Group Mixed-Effects Models (Subject Random Effects)
This table summarizes the results from the mixed-effects models applied to the case group data, accounting for subject-specific random effects.
Case Group Mixed-Effects Models: Time × Carbohydrate Effects with Subject Random Effects
|
Analyte
|
Effect
|
F-value
|
P-value
|
Significance
|
|
acetate
|
timepoint_hr
|
1481.9514
|
0.0000
|
***
|
|
acetate
|
carbohydrate_type
|
4.3256
|
0.0199
|
|
|
acetate
|
timepoint_hr:carbohydrate_type
|
4.4140
|
0.0185
|
|
|
butyrate
|
timepoint_hr
|
206.1035
|
0.0000
|
***
|
|
butyrate
|
carbohydrate_type
|
1.0672
|
0.3536
|
ns
|
|
butyrate
|
timepoint_hr:carbohydrate_type
|
1.0650
|
0.3543
|
ns
|
|
propionate
|
timepoint_hr
|
281.7401
|
0.0000
|
***
|
|
propionate
|
carbohydrate_type
|
3.6395
|
0.0353
|
|
|
propionate
|
timepoint_hr:carbohydrate_type
|
3.2477
|
0.0493
|
|
|
5aminovalerate
|
timepoint_hr
|
107.8313
|
0.0000
|
***
|
|
5aminovalerate
|
carbohydrate_type
|
0.1321
|
0.8766
|
ns
|
|
5aminovalerate
|
timepoint_hr:carbohydrate_type
|
0.1218
|
0.8857
|
ns
|
|
succinate
|
timepoint_hr
|
32.6569
|
0.0000
|
***
|
|
succinate
|
carbohydrate_type
|
1.5134
|
0.2325
|
ns
|
|
succinate
|
timepoint_hr:carbohydrate_type
|
1.3187
|
0.2789
|
ns
|
Delta Change Analysis (48h vs 0h)
To further investigate the magnitude of change over time, we calculated the delta (change) in concentration for each analyte between 48h and 0h. This approach focuses on the response magnitude and allows us to test whether the group or carbohydrate type influences how strongly subjects respond to the intervention over time.
Delta Summary Statistics
This table provides summary statistics for the calculated delta values (48h - 0h), showing the mean change and variability.
Summary Statistics for Delta Change (48h - 0h)
|
Group
|
Carbohydrate Type
|
Analyte
|
n
|
Mean Delta
|
SD Delta
|
SEM Delta
|
|
control
|
no_carbohydrate
|
5aminovalerate
|
8
|
0.967
|
0.464
|
0.164
|
|
control
|
no_carbohydrate
|
acetate
|
8
|
26.064
|
8.414
|
2.975
|
|
control
|
no_carbohydrate
|
butyrate
|
8
|
4.857
|
1.947
|
0.688
|
|
control
|
no_carbohydrate
|
propionate
|
8
|
5.263
|
1.467
|
0.519
|
|
control
|
no_carbohydrate
|
succinate
|
8
|
1.279
|
1.171
|
0.414
|
|
control
|
rapid_digestible
|
5aminovalerate
|
8
|
0.797
|
0.254
|
0.090
|
|
control
|
rapid_digestible
|
acetate
|
8
|
31.260
|
6.899
|
2.439
|
|
control
|
rapid_digestible
|
butyrate
|
8
|
7.312
|
4.161
|
1.471
|
|
control
|
rapid_digestible
|
propionate
|
8
|
3.411
|
1.818
|
0.643
|
|
control
|
rapid_digestible
|
succinate
|
8
|
1.510
|
2.004
|
0.708
|
|
control
|
slow_digestible
|
5aminovalerate
|
8
|
0.896
|
0.279
|
0.099
|
|
control
|
slow_digestible
|
acetate
|
8
|
32.358
|
8.054
|
2.847
|
|
control
|
slow_digestible
|
butyrate
|
8
|
8.556
|
3.313
|
1.171
|
|
control
|
slow_digestible
|
propionate
|
8
|
4.650
|
1.914
|
0.677
|
|
control
|
slow_digestible
|
succinate
|
8
|
1.962
|
2.262
|
0.800
|
|
case
|
no_carbohydrate
|
5aminovalerate
|
8
|
0.894
|
0.389
|
0.138
|
|
case
|
no_carbohydrate
|
acetate
|
8
|
29.969
|
3.483
|
1.231
|
|
case
|
no_carbohydrate
|
butyrate
|
8
|
5.723
|
1.141
|
0.404
|
|
case
|
no_carbohydrate
|
propionate
|
8
|
6.043
|
1.889
|
0.668
|
|
case
|
no_carbohydrate
|
succinate
|
8
|
0.846
|
1.101
|
0.389
|
|
case
|
rapid_digestible
|
5aminovalerate
|
8
|
0.811
|
0.570
|
0.201
|
|
case
|
rapid_digestible
|
acetate
|
8
|
31.058
|
6.913
|
2.444
|
|
case
|
rapid_digestible
|
butyrate
|
8
|
6.530
|
3.767
|
1.332
|
|
case
|
rapid_digestible
|
propionate
|
8
|
4.156
|
1.555
|
0.550
|
|
case
|
rapid_digestible
|
succinate
|
8
|
1.405
|
1.546
|
0.547
|
|
case
|
slow_digestible
|
5aminovalerate
|
8
|
0.807
|
0.582
|
0.206
|
|
case
|
slow_digestible
|
acetate
|
8
|
35.708
|
5.296
|
1.872
|
|
case
|
slow_digestible
|
butyrate
|
8
|
7.350
|
2.300
|
0.813
|
|
case
|
slow_digestible
|
propionate
|
8
|
5.037
|
2.360
|
0.834
|
|
case
|
slow_digestible
|
succinate
|
8
|
1.772
|
1.786
|
0.631
|
Delta Group Comparisons
This table shows the results of t-tests comparing the delta values between the control and case groups.
Group Comparisons of Delta Change (Response Magnitude)
|
analyte
|
.y.
|
group1
|
group2
|
n1
|
n2
|
statistic
|
df
|
p
|
p.adj
|
p.adj.signif
|
|
5aminovalerate
|
delta_48h_0h
|
control
|
case
|
24
|
24
|
0.4030
|
40.3837
|
0.689
|
0.6890
|
ns
|
|
acetate
|
delta_48h_0h
|
control
|
case
|
24
|
24
|
-1.1701
|
41.8753
|
0.249
|
0.6525
|
ns
|
|
butyrate
|
delta_48h_0h
|
control
|
case
|
24
|
24
|
0.4200
|
42.5161
|
0.677
|
0.6890
|
ns
|
|
propionate
|
delta_48h_0h
|
control
|
case
|
24
|
24
|
-1.1375
|
45.5565
|
0.261
|
0.6525
|
ns
|
|
succinate
|
delta_48h_0h
|
control
|
case
|
24
|
24
|
0.5079
|
44.3472
|
0.614
|
0.6890
|
ns
|
Delta Carbohydrate Comparisons
This table presents the results of ANOVA tests on the delta values to examine the effect of carbohydrate type on the magnitude of change.
ANOVA Results for Carbohydrate Effects on Delta Change
|
analyte
|
Effect
|
DFn
|
DFd
|
F
|
p
|
p<.05
|
ges
|
p.adj
|
p.adj.signif
|
|
5aminovalerate
|
carbohydrate_type
|
2
|
45
|
0.355
|
0.703
|
|
0.016
|
0.703
|
ns
|
|
acetate
|
carbohydrate_type
|
2
|
45
|
3.253
|
0.048
|
|
0.126
|
0.080
|
ns
|
|
butyrate
|
carbohydrate_type
|
2
|
45
|
3.404
|
0.042
|
|
0.131
|
0.080
|
ns
|
|
propionate
|
carbohydrate_type
|
2
|
45
|
4.218
|
0.021
|
|
0.158
|
0.080
|
ns
|
|
succinate
|
carbohydrate_type
|
2
|
45
|
0.956
|
0.392
|
|
0.041
|
0.490
|
ns
|
Delta Carbohydrate Post-Hoc Comparisons
Following the ANOVA on delta values, pairwise t-tests were performed to compare each carbohydrate type to the ‘no carbohydrate’ control. The results are shown below.
Pairwise Comparisons of Delta Change vs No Carbohydrate
|
analyte
|
.y.
|
group1
|
group2
|
n1
|
n2
|
p
|
p.signif
|
p.adj
|
p.adj.signif
|
|
5aminovalerate
|
delta_48h_0h
|
no_carbohydrate
|
rapid_digestible
|
16
|
16
|
0.4090
|
ns
|
0.5112
|
ns
|
|
5aminovalerate
|
delta_48h_0h
|
no_carbohydrate
|
slow_digestible
|
16
|
16
|
0.6060
|
ns
|
0.6060
|
ns
|
|
acetate
|
delta_48h_0h
|
no_carbohydrate
|
rapid_digestible
|
16
|
16
|
0.1900
|
ns
|
0.3100
|
ns
|
|
acetate
|
delta_48h_0h
|
no_carbohydrate
|
slow_digestible
|
16
|
16
|
0.0143
|
|
0.0477
|
|
|
butyrate
|
delta_48h_0h
|
no_carbohydrate
|
rapid_digestible
|
16
|
16
|
0.1200
|
ns
|
0.3000
|
ns
|
|
butyrate
|
delta_48h_0h
|
no_carbohydrate
|
slow_digestible
|
16
|
16
|
0.0130
|
|
0.0477
|
|
|
propionate
|
delta_48h_0h
|
no_carbohydrate
|
rapid_digestible
|
16
|
16
|
0.0058
|
**
|
0.0477
|
|
|
propionate
|
delta_48h_0h
|
no_carbohydrate
|
slow_digestible
|
16
|
16
|
0.2170
|
ns
|
0.3100
|
ns
|
|
succinate
|
delta_48h_0h
|
no_carbohydrate
|
rapid_digestible
|
16
|
16
|
0.5010
|
ns
|
0.5567
|
ns
|
|
succinate
|
delta_48h_0h
|
no_carbohydrate
|
slow_digestible
|
16
|
16
|
0.1740
|
ns
|
0.3100
|
ns
|
Delta Interaction Analysis
To assess the combined effects of group and carbohydrate type on the delta values, a two-way ANOVA was conducted. The results are summarized in this table.
Two-way ANOVA on Delta Change: Group × Carbohydrate Interactions
|
Analyte
|
Effect
|
P-value
|
Adjusted P-value
|
Significance
|
|
5aminovalerate
|
group
|
0.699
|
0.8975
|
ns
|
|
5aminovalerate
|
carbohydrate_type
|
0.718
|
0.8975
|
ns
|
|
5aminovalerate
|
group:carbohydrate_type
|
0.939
|
0.9610
|
ns
|
|
acetate
|
group
|
0.233
|
0.7230
|
ns
|
|
acetate
|
carbohydrate_type
|
0.051
|
0.2550
|
ns
|
|
acetate
|
group:carbohydrate_type
|
0.647
|
0.8975
|
ns
|
|
butyrate
|
group
|
0.665
|
0.8975
|
ns
|
|
butyrate
|
carbohydrate_type
|
0.048
|
0.2550
|
ns
|
|
butyrate
|
group:carbohydrate_type
|
0.584
|
0.8975
|
ns
|
|
propionate
|
group
|
0.241
|
0.7230
|
ns
|
|
propionate
|
carbohydrate_type
|
0.024
|
0.2550
|
ns
|
|
propionate
|
group:carbohydrate_type
|
0.947
|
0.9610
|
ns
|
|
succinate
|
group
|
0.622
|
0.8975
|
ns
|
|
succinate
|
carbohydrate_type
|
0.414
|
0.8975
|
ns
|
|
succinate
|
group:carbohydrate_type
|
0.961
|
0.9610
|
ns
|
Delta Mixed-Effects Models
This table summarizes the results from the mixed-effects models applied to the delta values, accounting for subject-specific random effects.
Delta Change Mixed-Effects Models: Group × Carbohydrate Effects
|
Analyte
|
Effect
|
F-value
|
P-value
|
Significance
|
|
acetate
|
group
|
0.6260
|
0.4404
|
ns
|
|
acetate
|
carbohydrate_type
|
22.7732
|
0.0000
|
***
|
|
acetate
|
group:carbohydrate_type
|
3.1210
|
0.0578
|
ns
|
|
butyrate
|
group
|
0.0965
|
0.7601
|
ns
|
|
butyrate
|
carbohydrate_type
|
10.0422
|
0.0004
|
***
|
|
butyrate
|
group:carbohydrate_type
|
1.6684
|
0.2045
|
ns
|
|
propionate
|
group
|
0.6049
|
0.4481
|
ns
|
|
propionate
|
carbohydrate_type
|
28.3790
|
0.0000
|
***
|
|
propionate
|
group:carbohydrate_type
|
0.3822
|
0.6854
|
ns
|
|
5aminovalerate
|
group
|
0.0709
|
0.7935
|
ns
|
|
5aminovalerate
|
carbohydrate_type
|
1.3520
|
0.2731
|
ns
|
|
5aminovalerate
|
group:carbohydrate_type
|
0.2555
|
0.7761
|
ns
|
|
succinate
|
group
|
0.1096
|
0.7449
|
ns
|
|
succinate
|
carbohydrate_type
|
4.7456
|
0.0157
|
|
|
succinate
|
group:carbohydrate_type
|
0.2126
|
0.8096
|
ns
|
Mixed-Effects Model Results
To account for the repeated measures design and the variability between subjects, we utilized linear mixed-effects models. These models include subject as a random effect, allowing us to more accurately assess the fixed effects of group, carbohydrate type, and time.
Mixed-Effects Model Summary
This table presents a summary of the full mixed-effects models, including F-values and p-values for all fixed effects.
Mixed-Effects Models: F-values and P-values for Fixed Effects
|
Analyte
|
Effect
|
F-value
|
P-value
|
Significance
|
|
acetate
|
group
|
0.5326
|
0.4761
|
ns
|
|
acetate
|
carbohydrate_type
|
7.1680
|
0.0014
|
**
|
|
acetate
|
timepoint_hr
|
1479.2346
|
0.0000
|
***
|
|
acetate
|
group:carbohydrate_type
|
1.7167
|
0.1862
|
ns
|
|
acetate
|
group:timepoint_hr
|
2.1173
|
0.1496
|
ns
|
|
acetate
|
carbohydrate_type:timepoint_hr
|
4.6260
|
0.0126
|
|
|
acetate
|
group:carbohydrate_type:timepoint_hr
|
0.6340
|
0.5331
|
ns
|
|
butyrate
|
group
|
1.4101
|
0.2524
|
ns
|
|
butyrate
|
carbohydrate_type
|
4.8623
|
0.0102
|
|
|
butyrate
|
timepoint_hr
|
373.5928
|
0.0000
|
***
|
|
butyrate
|
group:carbohydrate_type
|
0.8111
|
0.4480
|
ns
|
|
butyrate
|
group:timepoint_hr
|
0.2889
|
0.5924
|
ns
|
|
butyrate
|
carbohydrate_type:timepoint_hr
|
4.9696
|
0.0092
|
**
|
|
butyrate
|
group:carbohydrate_type:timepoint_hr
|
0.8256
|
0.4417
|
ns
|
|
propionate
|
group
|
0.0015
|
0.9697
|
ns
|
|
propionate
|
carbohydrate_type
|
7.3837
|
0.0011
|
**
|
|
propionate
|
timepoint_hr
|
539.7055
|
0.0000
|
***
|
|
propionate
|
group:carbohydrate_type
|
0.0689
|
0.9335
|
ns
|
|
propionate
|
group:timepoint_hr
|
2.4183
|
0.1239
|
ns
|
|
propionate
|
carbohydrate_type:timepoint_hr
|
6.9780
|
0.0016
|
**
|
|
propionate
|
group:carbohydrate_type:timepoint_hr
|
0.0940
|
0.9104
|
ns
|
|
5aminovalerate
|
group
|
0.4662
|
0.5045
|
ns
|
|
5aminovalerate
|
carbohydrate_type
|
0.7003
|
0.4995
|
ns
|
|
5aminovalerate
|
timepoint_hr
|
296.3213
|
0.0000
|
***
|
|
5aminovalerate
|
group:carbohydrate_type
|
0.1157
|
0.8909
|
ns
|
|
5aminovalerate
|
group:timepoint_hr
|
0.2449
|
0.6220
|
ns
|
|
5aminovalerate
|
carbohydrate_type:timepoint_hr
|
0.5405
|
0.5846
|
ns
|
|
5aminovalerate
|
group:carbohydrate_type:timepoint_hr
|
0.1021
|
0.9030
|
ns
|
|
succinate
|
group
|
1.0964
|
0.3106
|
ns
|
|
succinate
|
carbohydrate_type
|
2.9249
|
0.0594
|
ns
|
|
succinate
|
timepoint_hr
|
53.4997
|
0.0000
|
***
|
|
succinate
|
group:carbohydrate_type
|
0.0777
|
0.9253
|
ns
|
|
succinate
|
group:timepoint_hr
|
0.3696
|
0.5449
|
ns
|
|
succinate
|
carbohydrate_type:timepoint_hr
|
1.3509
|
0.2649
|
ns
|
|
succinate
|
group:carbohydrate_type:timepoint_hr
|
0.0605
|
0.9413
|
ns
|
Visualizations
To visually explore the data, we generated a series of plots. These visualizations illustrate the relationships between SCFA concentrations and the experimental variables, including group, carbohydrate type, and time. Each plot is designed to highlight different aspects of the data, from overall trends to individual subject responses.
SCFA Concentrations by Group and Carbohydrate Type

SCFA Concentrations by Carbohydrate Type and Time Point

Time Series Analysis

Concentration Heatmap

Interaction Effects

Subject-Level Individual Response Heatmap

Individual Subject Trajectories by Carbohydrate Source
